Powder needleless injection (PNI) is a novel method for administration of drugs, especially proteins and peptides. In this study, fluoresceinsodium was selected as model drug to evaluate and optimize the bioavailability of PNI on rabbits. The determination method was HPLC-FLD. The analyzing time of the method was within 3 minutes. A linear relationship of fluoresceinsodium was good between 0.0047 and 0.1872 microg x mL(-1) and the recovery of the method was larger than 97.6% (RSD < 3.6%). With the orthogonal experiments, the influence on PNI of several factors was compared. The model of the injector was the most important factor to enhance the bioavailability while gas pressure and drug diameter had positive correlation with both bioavailability and damage on skin. A condition of PNI, injector model C3, gas pressure of 4 MPa and drug diameter of 90-125 microm, was carried out and the bioavailability of self-designed PNI system was 34.9% in compare of the result (33%) of foreign correspording product in some degree. However, the mainly applicable drug types of PNI are proteins and peptides which is of high cost. So the self-designed PNI system needs to be improved.